Leptospira vaccine antigens for the prevention of Leptospirosis

ABSTRACT

Four antigenic preparations are provided, each of which contains a different protein from Leptospira which can be used immunologically in vaccines for leptospirosis caused by this organism. Also provided in the invention are polynucleotides encoding these four proteins and antibodies which bind the proteins for use in the diagnosis of leptospirosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 09/461,920, filed Dec.15, 1999, which is now U.S. Pat. No. 6,482,924, issued Nov. 19, 2002,which claims the benefit of priority to U.S. Ser. No. 60/113,288, filedDec. 22, 1998, both of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to antigenic preparations andspecifically to four Leptospira membrane proteins i.e., kinase,permease, mannosyltransferase and endoflagellin which are used to inducea protective immune response in animals. Such proteins can be usedimmunologically as vaccines for leptospirosis caused by this organism.Alternatively, diagnosis of leptospirosis can be performed by detectingthe presence of these proteins, antibody to the proteins, orpolynucleotides which encode the proteins.

BACKGROUND OF THE INVENTION

The pathogenic species of the Leptospira genus are the causative agentsof leptospirosis, a zoonotic disease of worldwide importance. Thebacterium is a gram negative spirochete which thrives under aerobicconditions. These bacteria have fastidious nutritional requirements andare able to utilize long chain fatty acids as a sole source of carbon.Leptospires are motile and their rapid, corkscrew motility serves as adistinguishing identifying feature of this organism. Leptospires areresistant to both metronidazole and 5-fluorouracil, and demonstrate ageneration time of 10 to 12 hours in vitro.

All pathogenic Leptospires were formerly classified as Leptospirainterrogans. Recently DNA homology studies have led to thereclassification of Leptospira interrogans into seven pathogenicLeptospira species: L. borgpetersenii sv hardjobovis, L. inadai, L.interrogans, L. kirshneri, L. noguchii, L. santarosai and L. weilli. Theserology of pathogenic Leptospira species which are responsible forLeptospirosis disease indicates that there are more than two hundredserovars (“sv”) within twenty three serogroups (Farr, R. W. Clin.Infect. Dis. 21:1-8 (1995)). There are many serovars responsible forleptospirosis disease worldwide. Leptospira interrogans sv pomona is acommon isolate from infected swine where it causes fever, jaundice,hemoglobinuria, and renal failure. Leptospira interrogans sv hardjobovisis an important cause of bovine disease, where it causes abortion andagalactia, and poses a zoonotic threat to humans after prolongedexposure to infected cattle. Many infected animal are asymptomatic.These animals can however act as carriers and shed leptospires throughurine.

Leptospira infections in cattle demonstrate a 16% infection rate, withrates being higher in beef cattle than in dairy cattle. The infectionrate is also higher in bulls than in cows. There is also a markedprevalence of certain serovars, which cause bovine leptospirosis. In arecent survey of cattle in the 48 states in the USA, of those animalspositive for leptospira, 84% were infected with sv hardiobovis, 12% wereinfected with sv pomona, and 4% were infected with sv griptotyphosa(Miller, D. A. et al., Am. J. Vet. res. 52(11):1761-1765 (1991)).

Little is known concerning the pathogenesis of Leptospira infections.Infections are usually transmitted by contact with urine from aninfected animal. Soil and water which has been contaminated withinfected urine can also transmit infection, although the prolongedsurvivability of leptospires under these conditions is questionable.Survival of Leptospira outside the host is fostered by a temperature of22° C. or above, moisture, and a neutral to slightly alkalineenvironment. Leptospira are readily killed by temperatures above 60° C.,detergents, desiccation, and acidity. Once the leptospira has invadedthe host, attachment to and penetration of the intercellular junctionsof mucosal epithelial cells is a crucial step in the infection. Abacteremia usually results and is the first pathological conditionassociated with leptospirosis. Once in circulation, the bacterium cancolonize the kidneys, where they may cause an acute or chronicinfection. Some potential virulence factors involved in the pathogenesisof leptospira are, hyaluronidase, urease, haemolysins, andphospholipases.

Leptospirosis is caused by pathogenic strains of Leptospira which arecapable of infecting most mammalian species. The predominant naturalreservoirs of pathogenic Leptospira are wild mammals, although othervertebrates occasionally are infected. In addition, several species ofleptospires are known pathogens of marine mammals, including Pacificharbor seals (Stampler, M. A. et al., J. Wild. Dis. 34(2):407-410(1989)). Domestic animals such as dogs, cattle, swine, sheep, goats andhorses, also may be major sources of human infections. Infection occurseither through direct contact with an infected animal or indirectcontact with contaminated soil or water. In livestock, the diseasecauses economic losses due to abortion, stillbirth, infertility,decreased milk production, and death.

The severity of human leprospirosis varies greatly and is determined toa large extent by the infecting strain and by the general health of thehost. The improved ability of regional laboratories to group Leptospirahas resulted in the recognition of the large number of serovars endemicin the United States, as well as the extent of infections in a varietyof animal species. Nevertheless, it is an infrequently diagnosed humandisease. Approximately 100 cases are reported annually in the UnitedStates.

Because of its prevalence in rodents and domestic animals, leptospirosishas been primarily a disease of person in occupations heavily exposed toanimals and animal products, such as sewer workers, swineherders,veterinarians, abattoir workers, and farmers (Vinetz, J. M., Cur. Opin.Infect. Dis. 10:357-361 (1997)). Also at risk are persons living inrodent-infested housing, such as urban slums, and dog owners. There is ahigher incidence in males. At present, the majority of cases occur inthe summer and fall in teenagers and young adults. Avocational exposureis now increasingly common.

Common source outbreaks attributed to contaminated ponds or slowlymoving streams are numerous. A high attack rate, summer season, youngage group, and the proximity of animals to the water typify most ofthese outbreaks. In some areas of the world, the runoff during floodingalso is highly infectious.

Sporadic disease may be acquired by direct contact with infectedanimals. Vaccination of domestic animals, which prevents clinicaldisease, may fail to prevent shedding of Leptospira. Pet dogs have beena prominent source of sporadic human cases. The convoluted renal tubulesof animal reservoirs harbor viable Leptospira, which are passed in theurine. The duration of asymptomatic urinary shedding varies with theanimal species; humans rarely shed Leptospira longer than a few months.

Forms of transmission other than direct and indirect contact withcontaminated urine are rare. Lactating animals shed Leptospira in themilk, but whole milk is leptospirocidal after a few hours, and no knownhuman cases have occurred in this manner. Leptospira are not shed insaliva, and animal bites are therefore not a direct source of infection.

The pathogenesis of leptospirosis is very similar to that of otherspirochetal diseases, including syphilis (caused by Treponema pallidum)and Lymeborreliosis (caused by Borrelia burgdoferi). Both syphilis andLyme borreliosis are are characterized by widespread dissemination earlyin the course of disease, including invasion of the central nervoussystem. Leptospira share this ability with other pathogenic spirochetessuch that meningitis is a common manifestation of leptospirosis. Anotherfeature of spirochetal infections is the ability to persist chronicallyin the host, as manifested in cases of tertiary syphilis and chronicLyme arthritis. (For a comprehensive review, see Baranton, G. and Old,I. G., Bull. Inst. Pasteur 93:63-95 (1995)).

Efforts to control leptospirosis have been hampered because virulentleptospires have the capacity for both long-term survival in theenvironment as well as persistent infection and shedding by wildlife andlivestock.

Leptospira membrane proteins are of great importance because they play akey role in bacterial pathogenesis. The identification of membraneproteins involved in Leptospira pathogenesis is significant tounderstanding not only leptospiral membrane proteins and theirinvolvement in pathogenesis, but also to understanding other spirochetalmembrane proteins and their role in pathogenesis.

Currently available leptospiral vaccines produce short-term immunity anddo not provide cross-protection against many of the 170 serovars ofpathogenic Leptospira (Thiermann, et al., J. Am. Vet. Med. Assoc.184:722 (1984)). These vaccines consist of inactivated whole organismsor outer envelope preparations which produce seroreactivity asdetermined by microscopic agglutination of intact organisms. The natureof the protective immunogens in these vaccine preparations has not beenconclusively elucidated, although several lines of evidence suggest thatlipopolysaccharide-like substance (LLS) may confer a degree ofprotection.

In terms of treatment of active infection, oxytetracycline is the drugof choice and is used routinely in the field to both cure infection andcarriage. Several vaccine preparations using bacterins or components oflipopolysaccharide have been used with variable success. Protection withthe current vaccines tend to be serovar specific and lack the ability togenerate a reproducible degree of protection.

SUMMARY OF THE INVENTION

The present invention is based on the identification of four Leptospiramembrane proteins i.e., kinase, permease, mannosyltransferase andendoflagellin, which are associated with pathogenic strains ofLeptospira. Due to spirochetal membrane fragility and the fact thatmembrane proteins are present in small amounts, there have been limiteddefinitive reports of membrane spanning spirochetal membrane proteinsuntil the present invention. The identification of in vivo expressedgenes by mRNA subtractive hybridization is a powerful means by which toidentify virulence-related genes. The present invention describes theidentification of three Leptospira interrogans sv pomona genes which areexpressed during colonization of the liver of infected Syrian hamsters.The present invention also describes a fourth gene identified from L.hardjobovis using a ZAP expression library. The invention also describesfour membrane proteins from Leptospira which are immunogenic and usefulfor inducing an immune response to pathogenic Leptospira as well asproviding a diagnostic target for leptospirosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. The complete nucleotide sequence, SEQ ID NO: 5, (FIG.1A) and deduced amino acid sequence, SEQ ID NO: 1, (FIG. 1B) ofLeptospira membrane protein kinase, (ORF1) from pHLE011 and pMW43. Theinitiation and termination codons are underlined in bold.

FIGS. 2A and 2B. The complete nucleotide sequence, SEQ ID NO: 6, (FIG.2A) and deduced amino acid sequence, SEQ ID NO: 2, (FIG. 2B) ofLeptospira membrane protein permease, (ORF2) from pHLE011 and pMW310.The initiation and termination codons are underlined and in bold.

FIGS. 3A and 3B. The complete nucleotide sequence, SEQ ID NO: 7, (FIG.3A) and deduced amino acid sequence, SEQ ID NO: 3, (FIG. 3B) ofLeptospira membrane protein mannosyltransferase, (ORF3) from pMW50. Theinitiation and termination codons are underlined and in bold.

FIGS. 4A and 4B. The complete nucleotide sequence, SEQ ID NO: 8 (FIG.4A) and deduced amino acid sequence, SEQ ID NO: 8, (FIG. 4B) ofLeptospira membrane protein endoflagellin from pDFX210. The initiationand termination codons are underlined and in bold.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides four immunogenic proteins from themembrane of a pathogenic Leptospira species. These membrane proteins arekinase, permease, mannosyltransferase and endoflagellin. Also includedare four polynucleotide sequences which encode these proteins.

The four immunogenic proteins of the present invention are useful inpharmaceutical compositions for inducing an immune response topathogenic Leptospira.

The invention includes a method of producing these membranes proteins ofLeptospira using recombinant DNA techniques. The genes for the fourmembrane proteins are cloned into plasmid vectors which are then used totransform E. coli.

The bacterial genes for these four membrane proteins can likely bederived from any strain of pathogenic Leptospira. Preferably theproteins are from Leptospira interrogans sv pomaona or Leptospiraborgpetersenii sv hardjobovis. These Leptospira organisms are publicallyavailable through the ATOC (10801 University Boulevard, Manassas, Va.,20220-2209), for example.

The invention provides polynucleotides encoding the four Leptospiramembrane proteins i.e. kinase, permease, mannosyltransferase andendoflagellin. These polynucleotides include DNA and RNA sequences whichencode these four proteins. It is understood that all polynucleotidesencoding all or a portion of these four proteins are also includedherein, so long as these polynucleotides encode polypeptides thatexhibit the function of the native or full length proteins, such as theability to induce or bind antibody. Such polynucleotides include bothnaturally occurring and intentionally manipulated, for example,mutagenized polynucleotides.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization procedures whichare well known in the art. These include, but are not limited to: 1)hybridization of probes to genomic libraries to detect shared nucleotidesequences and 2) antibody screening of expression libraries to detectshared structural features.

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereeach probe is potentially the complete complement of a specific DNAsequence in the hybridization sample which includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. By using stringent hybridizationconditions directed to avoid non-specific binding, it is possible, forexample, to allow the autoradiographic visualization of a specific DNAclone by the hybridization of the target DNA to that single probe in themixture which is its complete complement (Wallace, et al., Nucleic AcidResearch, 9:879(1981)).

Alternatively, an expression library can be screened indirectly for thefour membrane proteins of the invention having at least one epitope perprotein using antibodies to these proteins. Such antibodies can beeither polyclonally or monoclonally derived and used to detectexpression product indicative of the presence of Leptospira kinase,permease, mannosyltransferase and endoflagellin DNA. Generally, a lambdagt11 library is constructed and screened immunologically according tothe method of Huynh, et al., (in DNA Cloning: A Practical Approach, D.M. Glover, ed.,1:49 (1985)).

The development of specific DNA sequences encoding each of the kinase,permease, mannosyltransferase and endoflagellin membrane proteins canalso be obtained by: (1) isolation of a double-stranded DNA sequencefrom the genomic DNA, and (2) chemical manufacture of a DNA sequence toprovide the necessary codons for the protein of interest.

The polymerase chain reaction (PCR) technique can be utilized to obtainor amplify the four individual Leptospira membrane proteins from anystrain of Leptopira for subsequent cloning and expression of cDNAsencoding these four proteins (e.g., see U.S. Pat. Nos. 4,683,202;4,683,195; 4,889,818; Gyllensten et al., Proc. Nat'l Acad. Sci. USA,85:7652-7656 (1988); Ochman et al., Genetics, 120:621-623 (1988) Trigliaet al., Nucl. Acids. Res.,16:8156 (1988); Frohman et al., Proc. Nat'lAcad. Sci. USA, 85:8998-9002 (1988); Loh et al., Science, 243:217-220(1989)). Similarly, the PCR technique can be routinely used by thoseskilled in the art, to generate polynucleotide fragments encodingportions of any of the four Leptospiral membrane proteins of the instantinvention.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the four Leptospira membraneproteins or fragments thereof coding sequences and appropriatetranscriptional/translational control signals. These methods include invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. See, for example, the techniquesdescribed in Maniatis et al., Molecular Cloning A Laboratory Manual,Cold Spring Harbor Laboratory, N.Y., Chapter 12 (1982).

A variety of host-expression vector systems may be utilized to expressthe four Leptospira membrane proteins or fragments thereof. Theseinclude but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing a coding sequence for a Leptospiramembrane protein or fragment thereof; yeast transformed with recombinantyeast expression vectors containing a coding sequence for a Leptospiramembrane protein or fragment thereof; insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing acoding sequence for a Leptospira membrane protein or fragment thereof;or animal cell systems infected with recombinant virus expressionvectors (e.g., adenovirus, vaccinia virus) containing a coding sequencefor a Leptospira membrane protein or fragment thereof.

The expression elements of these vectors vary in their strength andspecificities. Depending on the host/vector system utilized, any of anumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedrin promoter may beused; when cloning in mammalian cell systems, promoters such as theadenovirus late promoter or the vaccinia virus 7.5K promoter may beused. Promoters produced by recombinant DNA or synthetic techniques mayalso be used to provide for transcription of the inserted codingsequence for a Leptospira membrane protein or fragment thereof.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol. 2, Ed. Ausubel et al., Greene Publish. Assoc. & WileyInterscience Ch. 13 (1988); Grant et al., Expression and SecretionVectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, Acad.Press, N.Y., Vol. 153, pp. 516-544 (1987); Glover, DNA Cloning, Vol. II,IRL Press, Wash., D.C. Ch.3 (1986); and Bitter, Heterologous GeneExpression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad.Press, N.Y., Vol. 152, pp. 673-684 (1987); and The Molecular Biology ofthe Yeast Saccharomyces, Eds. Strathern et al., Cold Spring HarborPress, Vols. I and II (1982). For complementation assays in yeast, cDNAsfor Leptospira membrane proteins or fragments thereof may be cloned intoyeast episomal plasmids (YEp) which replicate autonomously in yeast dueto the presence of the yeast 2 mu circle. Any of the Leptospira membraneprotein or fragment thereof sequence may be cloned behind either aconstitutive yeast promoter such as ADH or LEU2 or an inducible promotersuch as GAL (Cloning in Yeast, Ch. 3, R. Rothstein In; DNA Cloning Vol.11, A Practical Approach, Ed. D M Glover, IRL Press, Wash., D.C.(1986)). Constructs may contain the 5′ and 3′ non-translated regions ofa cognate Leptospira membrane protein mRNA or those corresponding to ayeast gene. YEp plasmids transform at high efficiency and the plasmidsare extremely stable. Alternatively vectors may be used which promoteintegration of foreign DNA sequences into the yeast chromosome.

A particularly good expression system which could be used to express oneof the four Leptospira membrane proteins or fragments thereof is aninsect system. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The Leptospira membraneprotein or fragment thereof coding sequence may be cloned intonon-essential regions (for example the polyhedrin gene) of the virus andplaced under control of an AcNPV promoter (for example the polyhedrinpromoter). Successful insertion of the polyhedrin gene results inproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed. (e.g., see Smith et al., J. Biol., 46:586(1983); U.S. Pat. No. 4,215,051).

In cases where an adenovirus is used as an expression vector, theLeptospira membrane protein or fragment thereof coding sequence may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vivo or invitro recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing a Leptospira membrane protein offragment thereof in infected hosts. (e.g., See Logan & Shenk, Proc.Natl. Acad. Sci., (USA) 81:3655-3659 (1984)). Alternatively, thevaccinia 7.5K promoter may be used. (e.g., see Mackett et al., Proc.Natl. Acad. Sci., (USA) 79:7415-7419 (1982); Mackett et al., J. Virol.,49:857-864 (1984); Panicali et al., Proc. Natl. Acad. Sci., 79:4927-4931(1982)).

Specific initiation signals may also be required for efficienttranslation of the inserted Leptospira membrane protein or fragmentthereof coding sequences. These signals include the ATG initiation codonand adjacent sequences. In cases where the entire Leptospira membraneprotein genome, including its own initiation codon and adjacentsequences, are inserted into the appropriate expression vectors, noadditional translational control signals may be needed. However, incases where only a portion of the Leptospiral membrane protein codingsequence is inserted, exogenous translational control signals, includingthe ATG initiation codon, must be provided. Furthermore, the initiationcodon must be in phase with the reading frame of the Leptospiralmembrane protein or fragment thereof coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bitter et al., Methods in Enzymol.,153:516-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression driven by certainpromoters can be elevated in the presence of certain inducers, (e.g.,zinc and cadmium ions for metallothionein promoters). Therefore,expression of the genetically engineered Leptospiral membrane protein orfragment thereof may be controlled. This is important if the proteinproduct of the cloned foreign gene is lethal to host cells. Furthermore,modifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed.

The host cells which contain the Leptospira membrane protein or fragmentthereof coding sequence and which express the biologically activeLeptospira membrane protein or fragment thereof gene product may beidentified by at least four general approaches: (a) DNA-DNAhybridization; (b) the presence or absence of “marker” gene functions;(c) assessing the level of transcription as measured by expression of aLeptospiral membrane protein mRNA transcripts in host cells; and (d)detection of Leptospiral membrane protein gene products as measured byimmunoassays or by its biological activity.

In the first approach, the presence of the Leptospira membrane proteinor fragment thereof coding sequence inserted in the expression vectorcan be detected by DNA-DNA hybridization using probes comprisingnucleotide sequences that are homologous to the Leptospira membraneprotein coding sequence or particular portions thereof substantially asdescribed recently (Goeddert et al., 1988, Proc. Natl. Acad. Sci. USA,85:4051-4055).

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if the Leptospira membrane protein or fragment thereof coding sequenceis inserted within a marker gene sequence of the vector, recombinantscontaining the Leptospira membrane protein or fragment thereof codingsequence can be identified by the absence of the marker gene function.Alternatively, a marker gene can be placed in tandem with the Leptospiramembrane protein or fragment thereof coding sequence under the controlof the same or different promoter used to control the expression of theLeptospira membrane protein coding sequence. Expression of the marker inresponse to induction or selection indicates expression of theLeptospira membrane protein coding sequence.

In the third approach, transcriptional activity for the Leptospiramembrane protein or fragment thereof coding region can be assessed byhybridization assays. For example, RNA can be isolated and analyzed byNorthern blot using a probe homologous to the Leptospira membraneprotein or fragment thereof coding sequence or particular portionsthereof substantially as described (Goeddert et al., 1988, Proc. Natl.Acad. Sci. USA, 85:4051-4055). Alternatively, total nucleic acids of thehost cell may be extracted and assayed for hybridization to such probes.

In the fourth approach, the expression of the Leptospira membraneprotein or fragment thereof product can be assessed immunologically, forexample by Western blots, immunoassays such as radioimmunoprecipitation,enzyme-linked immunoassays and the like.

Once a recombinant that expresses a Leptospira membrane protein orfragment thereof is identified, the gene product should be analyzed.This can be achieved by assays based on the physical, immunological orfunctional properties of the product.

A Leptospira membrane protein or fragment thereof should beimmunoreactive whether it results from the expression of the entire genesequence, a portion of the gene sequence or from two or more genesequences which are ligated to direct the production of chimericproteins. This reactivity may be demonstrated by standard immunologicaltechniques, such as radioimmunoprecipitation, radioimmune competition,or immunoblots.

DNA sequences encoding the four membrane proteins of the invention canbe expressed in vitro by DNA transfer into a suitable host cell.“Recombinant host cells” or “host cells” are cells in which a vector canbe propagated and its DNA expressed. The term also includes any progenyof the subject host cell. It is understood that not all progeny areidentical to the parental cell since there may be mutations that occurat replication. However, such progeny are included when the terms aboveare used.

The term “host cell” as used in the present invention is meant toinclude not only prokaryotes, but also, such eukaryotes as yeasts,filamentous fungi, as well as plant and animal cells. The term“prokaryote” is meant to include all bacteria, which can be transformedwith the genes for the expression of the four Leptospira membraneproteins of the invention. Prokaryotic hosts may include Gram negativeas well as Gram positive bacteria, such as E. coil, S. typhimurium, andBacillus subtilis.

A recombinant DNA molecule coding for the four Leptospira membraneproteins of the invention can be used to transform a host using any ofthe techniques commonly known to those of ordinary skill in the art.Especially preferred is the use of a plasmid containing any of the fourLeptospira membrane protein coding sequences for purposes of prokaryotictransformation. Where the host is prokaryotic, such as E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method by procedures well known in the art. Alternatively,MgCl₂ or RbCl can be used. Transformation can also be performed afterforming a protoplast of the host cell.

In the present invention, any of the four Leptospira membrane proteinencoding sequences may be inserted into a recombinant expression vector.The term “recombinant expression vector” refers to a plasmid, virus orother vehicle known in the art that has been manipulated by insertion orincorporation of any of the four Leptospira membrane protein geneticsequences. Such expression vectors contain a promotor sequence whichfacilitates the efficient transcription of the inserted genetic sequencein the host. The expression vector typically contains an origin ofreplication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells. The transformedprokaryotic hosts can be cultured according to means known in the art toachieve optimal cell growth. Various shuttle vectors for the expressionof foreign genes in yeast have been reported (Heinemann, et al., Nature,340:205 (1989); Rose, et al., Gene, 60:237 (1987)). Biologicallyfunctional DNA vectors capable of expression and replication in a hostare known in the art. Such vectors are used to incorporate DNA sequencesof the invention.

Methods for preparing fused, operably linked genes and expressing themin bacteria are known and are shown, for example, in U.S. Pat. No.4,366,246 which is incorporated herein by reference. The geneticconstructs and methods described therein can be utilized for expressionof any of the four Leptospira membrane proteins in prokaryotic hosts.

Examples of promoters which can be used in the invention are: rec A,trp, lac, tac, and bacteriophage lambda p[R] or p[L]. Examples ofplasmids which can be used in the invention are listed in Maniatis, etal., (Molecular Cloning, Cold Spring Harbor Laboratories, 1982).

Antibodies provided in the present invention are immunoreactive with anyof the four Leptospira membrane proteins. Antibody which consistsessentially of pooled monoclonal antibodies with different epitopicspecificities, as well as distinct monoclonal antibody preparations areprovided. Monoclonal antibodies are made from antigen containingfragments of the protein by methods well known in the art (Kohler, etal., Nature, 256:495 (1975); Current Protocols in Molecular Biology,Ausubel, et al., ed., (1989)).

The term “antibody” as used in this invention includes intact moleculesas well as fragments thereof, such as Fab, F(ab′)2, and Fv which arecapable of binding the epitopic determinant. These antibody fragmentsretain some ability to selectively bind with its antigen or receptor andare defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference).

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Antibodies which bind to any of the four Leptospira membrane proteins ofthe invention can be prepared using an intact polypeptide or fragmentscontaining small peptides of interest as the immunizing antigen.

Any of the proteins or fragments thereof of SEQ ID NOS: 1-4 can also beproduced by chemical synthesis of the amino acid sequence of any ofthese four proteins (Goeddert et al., Proc. Natl. Acad. Sci. USA,85:4051-4055 (1988)), as predicted from the cloning and sequencing of acDNA coding for any of these four Leptospira membrane proteins. The fourLeptospira membrane proteins may be chemically synthesized usingstandard peptide synthesis methods known in the art. These methodsinclude a solid-phase method devised by R. Bruce Merrifield, (Ericksonand Merrifield, “Solid-Phase Peptide Synthesis”, in The Proteins, Volume2, H. Neurath & R. Hill (eds.) Academic Press, Inc., New York pp.255-257; Merrifield, “Solid phase synthesis”, Science, 242:341-347(1986)). In the solid-phase method, amino acids are added stepwise to agrowing peptide chain that is linked to an insoluble matrix, such aspolystyrene beads. A major advantage of this method is that the desiredproduct at each stage is bound to beads that can be rapidly filtered andwashed and thus the need to purify intermediates is obviated. All of thereactions are carried out in a single vessel, which eliminates lossesdue to repeated transfers of products. This solid phase method ofchemical peptide synthesis can readily be automated making it feasibleto routinely synthesize peptides containing about 50 residues in goodyield and purity (Stewart and Young, Solid Phase Peptide Synthesis, 2nded., Pierce Chemical Co. (1984); Tam et al., J. Am. Chem. Soc., 105:6442(1983)).

Any of the proteins or fragments thereof of SEQ ID NOS: 1-4 used toimmunize an animal can be derived from translated cDNA or chemicalsynthesis which can be conjugated to a carrier protein, if desired. Suchcommonly used carriers which are chemically coupled to the peptideinclude keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serumalbumin (BSA), and tetanus toxoid. The coupled peptide is then used toimmunize the animal (e.g., a mouse, a rat, or a rabbit).

If desired, polyclonal or monoclonal antibodies can be further purified,for example, by binding to and elution from a matrix to which any of thefour proteins or a fragment thereof of any of the four proteins to whichthe antibodies were raised is bound. Those of skill in the art will knowof various techniques common in the immunology arts for purificationand/or concentration of polyclonal antibodies, as well as monoclonalantibodies (See for example, Coligan, et al., Unit 9, Current Protocolsin Immunology, Wiley Interscience, 1991, incorporated by reference).

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

Minor modifications of primary amino acid sequence of any of the fourproteins of the invention may result in proteins which havesubstantially equivalent function compared to the native Leptospiraproteins described herein. Such modifications may be deliberate, as bysite-directed mutagenesis, or may be spontaneous. All proteins producedby these modifications are included herein as long as native functionexists i.e., binds to antibody specific to the any of the fourLeptospira membrane proteins.

Modifications of primary amino acid sequence of any of the four membraneproteins also include conservative variations. The term “conservativevariation” as used herein denotes the replacement of an amino acidresidue by another, biologically similar residue. Examples ofconservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acids, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies raised tothe substituted protein or fragment thereof also immunoreact with theunsubstituted protein or fragment thereof.

Isolation and purification of microbially expressed protein, onfragments thereof, provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.

The invention extends to any host modified according to the methodsdescribed, or modified by any other methods, commonly known to those ofordinary skill in the art, such as, for example, by transfer of geneticmaterial using a lysogenic phage, and which result in a prokaryoteexpressing any of the four Leptospira genes for membrane proteinskinase, permease, mannosyltransferase or endoflagellin. Prokaryotestransformed with any of the four Leptospira genes encoding the fourmembrane proteins of the invention are particularly useful for theproduction of polypeptides which can be used for the immunization of ananimal (e.g., a rabbit).

In one embodiment, the invention provides a pharmaceutical compositionuseful for inducing an immune response to pathogenic Leptospira in ananimal comprising an immunologically effective amount of any one of thefour Leptospira membrane proteins in a pharmaceutically acceptablecarrier. The term “immunogenically effective amount,” as used indescribing the invention, is meant to denote that amount of Leptospiraantigen which is necessary to induce in an animal the production of animmune response to Leptospira. The four Leptospira membrane proteins ofthe invention are particularly useful in sensitizing the immune systemof an animal such that, as one result, an immune response is producedwhich ameliorates the effect of Leptospira infection.

Any of the four Leptospira membrane proteins of the invention can beadministered parenterally by injection, rapid infusion, nasopharyngealabsorption, dermal absorption, and orally. Pharmaceutically acceptablecarrier preparations for parenteral administration include sterile oraqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Carriers for occlusive dressings can be used to increaseskin permeability and enhance antigen absorption. Liquid dosage formsfor oral administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable forms for suspending theliposomes include emulsions, suspensions, solutions, syrups, and elixirscontaining inert diluents commonly used in the art, such as purifiedwater. Besides the inert diluents, such compositions can also includeadjuvants, wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents.

It is also possible for the antigenic preparations containing any of thefour Leptospira membrane proteins of the invention to include anadjuvant. Adjuvants are substances that can be used to nonspecificallyaugment a specific immune response. Normally, the adjuvant and theantigen are mixed prior to presentation to the immune system, orpresented separately, but into the same site of the animal (including ahuman) being immunized. Adjuvants can be loosely divided into severalgroups based on their composition.

These groups include oil adjuvants (for example, Freund's Complete andIncomplete), mineral salts (for example, AlK(SO₄)2, AlNa(SO₄)2,AlNH4(SO₄), silica, alum, Al(OH)3, Ca₃ (PO₄)2, kaolin, and carbon),polynucleotides (for example, poly IC and poly AU acids), and certainnatural substances (for example, wax D from Mycobactetium tuberculosis,as well as substances found in Colynebacterium parvum, Bordetellapertussis, and members of the genus Brucella).

In another embodiment, a method of inducing an immune response topathogenic Leptospira in an animal (including a human) is provided. Manydifferent techniques exist for the timing of the immunizations when amultiple immunization regimen is utilized. It is possible to use theantigenic preparation of the invention more than once to increase thelevels and diversity of expression of the immune response of theimmunized animal. Typically, if multiple immunizations are given, theywill be spaced two to four weeks apart. Subjects in which an immuneresponse to Leptospira is desirable include swine, cattle and humans.

Generally, the dosage of any of the four Leptospira membrane proteins ofthe invention administered to an animal (including a human) will varydepending on such factors as age, condition, sex and extent of disease,if any, and other variables which can be adjusted by one of ordinaryskill in the art.

The antigenic preparations of the invention can be administered aseither single or multiple dosages and can vary from about 10 ug to about1,000 ug for any of the four Leptospira membrane protein antigen perdose, more preferably from about 50 ug to about 700 ug antigen per dose,most preferably from about 50 ug to about 300 ug antigen per dose. Whenused for immunotherapy, the monoclonal antibodies of the invention maybeunlabeled or labeled with a therapeutic agent. These agents can becoupled either directly or indirectly to the monoclonal antibodies ofthe invention. One example of indirect coupling is by use of a spacermoiety. These spacer moieties, in turn, can be either insoluble orsoluble (Diener, et al., Science, 231:148 (1986)) and can be selected toenable drug release from the monoclonal antibody molecule at the targetsite. Examples of therapeutic agents which can be coupled to themonoclonal antibodies of the invention for immunotherapy are drugs,radioisotopes, lectins, and toxins. The labeled or unlabeled monoclonalantibodies of the invention can also be used in combination withtherapeutic agents such as those described above.

Especially preferred are therapeutic combinations comprising themonoclonal antibody of the invention and immunomodulators and otherbiological response modifiers. When the monoclonal antibody of theinvention is used in combination with various therapeutic agents, suchas those described herein, the administration of the monoclonal antibodyand the therapeutic agent usually occurs substantiallycontemporaneously. The term “substantially contemporaneously” means thatthe monoclonal antibody and the therapeutic agent are administeredreasonably close together with respect to time. Usually, it is preferredto administer the therapeutic agent before the monoclonal antibody. Forexample, the therapeutic agent can be administered 1 to 6 days beforethe monoclonal antibody. The administration of the therapeutic agent canbe daily, or at any other interval, depending upon such factors, forexample, as the nature of the disorder, the condition of the patient andhalf-life of the agent.

The dosage ranges for the administration of monoclonal antibodies of theinvention are those large enough to produce the desired effect in whichthe onset symptoms of the leptospiral disease are ameliorated. Thedosage should not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the subject and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician in theevent of any complication. Dosage can vary from about 0.1 mg/kg to about2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in one ormore dose administrations daily, for one or several days. Generally,when the monoclonal antibodies of the invention are administeredconjugated with therapeutic agents, lower dosages, comparable to thoseused for in vivo diagnostic imaging, can be used.

The monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual perfusion over time. Themonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, or transdermally, alone or in combination with effectorcells.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents and inert gasesand the like.

In a further embodiment, the invention provides a method of detecting apathogenic Leptospira-associated disorder in an animal (including ahuman) comprising contacting a cell component with a reagent which bindsto the cell component. The cell component can be nucleic acid, such asDNA or RNA, or it can be protein. When the component is nucleic acid,the reagent is a nucleic acid probe or PCR primer. When the cellcomponent is protein, the reagent is an antibody probe.

The probes are detectably labeled, for example, with a radioisotope, afluorescent compound, a bioluminescent compound, a chemiluminescentcompound, a metal chelator or an enzyme. Those of ordinary skill in theart will know of other suitable labels for binding to the antibody, orwill be able to ascertain such, using routine experimentation.

For purposes of the invention, an antibody or nucleic acid probespecific for any of the four Leptospira proteins of the invention may beused to detect the presence of that protein (using antibody) orpolynucleotide (using nucleic acid probe) in biological fluids ortissues. Any specimen containing a detectable amount any of the fourLeptospira membrane protein antigens or polynucleotides can be used. Apreferred specimen of this invention is blood, urine, cerebrospinalfluid, or tissue of endothelial origin.

When the cell component is nucleic acid, it may be necessary to amplifythe nucleic acid prior to binding with a Leptospira specific probe.Preferably, polymerase chain reaction (PCR) is used, however, othernucleic acid amplification procedures such as ligase chain reaction(LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used.

Another technique which may also result in greater sensitivity consistsof coupling antibodies to low molecular weight haptens. These haptenscan then be specifically detected by means of a second reaction. Forexample, it is common to use such haptens as biotin, which reacts withavidin, or dinitrophenyl, pyridoxal, and fluorescein, which can reactwith specific antihapten antibodies.

Alternatively, any of the four Leptospira membrane proteins of theinvention can be used to detect antibodies to any one of those fourproteins in a specimen. The four Leptospira membrane proteins of theinvention are particularly suited for use in immunoassays in which itcan be utilized in liquid phase or bound to a solid phase carrier. Inaddition, any one of the four Leptospira membrane proteins used in theseassays can be detectably labeled in various ways.

Examples of immunoassays which can utilize any one of the four membraneproteins of the invention are competitive and noncompetitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA), the enzyme-linkedimmunosorbant assay, the sandwich (immunometric) assay, a precipitinreaction, a gel immunodiffusion assay, an agglutination assay, afluorescent immunoassay, a protein A immunoassay and animmunoelectrophoresis assay, and the Western blot assay. Detection ofantibodies which bind to any one of the four Leptospira membraneproteins of the invention can be done utilizing immunoassays which runin either the forward, reverse, or simultaneous modes, includingimmunohistochemical assays on physiological samples. The concentrationof the Leptopira membrane protein which is used will vary depending onthe type of immunoassay and nature of the detectable label which isused. However, regardless of the type of immunoassay which is used, theconcentration of the Leptospira membrane protein utilized can be readilydetermined by one of ordinary skill in the art using routineexperimentation.

Any one of the four Leptospira membrane proteins of the invention can bebound to many different carriers and used to detect the presence ofantibody specifically reactive with the protein.

Examples of well-known carriers include glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, polycarbonate, dextran, nylon,amyloses, naturaland modified celluloses, polyacrylamides, agaroses, andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention.

Those skilled in the art will know of other suitable carriers forbinding any one of the four Leptospira membrane proteins of theinvention or will be able to ascertain such, using routineexperimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,colloidal metals, fluorescent compounds, chemiluminescent compounds, andbioluminescent compounds.

For purposes of the invention, the antibody which binds to any one ofthe Leptospira membrane proteins of the invention may be present invarious biological fluids and tissues. Any sample containing adetectable amount of antibodies to any one of the four Leptospiramembrane proteins can be used. Normally, a sample is a liquid such asurine, saliva, cerebrospinal fluid, blood, serum and the like, or asolid or semi-solid such as tissue, feces and the like. The monoclonalantibodies of the invention, directed toward any one of the fourLeptospira membrane proteins of the invention, are also useful for thein vivo. detection of antigen. The delectably labeled monoclonalantibody is given in a dose which is diagnostically effective. The term“diagnostically effective” means that the amount of delectably labeledmonoclonal antibody is administered in sufficient quantity to enabledetection of any one of the four Leptospira membrane protein antigensfor which the monoclonal antibodies are specific.

The concentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to those cells,body fluid, or tissue having any one of the four Leptospira membraneproteins is detectable compared to the background. Further, it isdesirable that the detectably labeled monoclonal antibody be rapidlycleared from the circulatory system in order to give the besttarget-to-background signal ratio.

As a rule, the dosage of detectably labeled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the animal (including a human). The dosage ofmonoclonal antibody can vary from about 0.001 mg/m² to about 500 mg/m²,preferably 0.1 mg/m² to about 200 mg/m², most preferably about 0.1 mg/m²to about 10 mg/m². Such dosages may vary, for example, depending onwhether multiple injections are given, and other factors known to thoseof skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 key range, which may be readilydetected by conventional gamma cameras.

For in vivo diagnosis, radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr and²⁰¹TI.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

The monoclonal antibodies of the invention can be used to monitor thecourse of amelioration of Leptospira associated disorder. Thus, bymeasuring the increase or decrease of any one of the four Leptospiramembrane proteins of the invention or antibodies to the any one of thefour Leptospira membrane proteins present in various body fluids ortissues, it would be possible to determine whether a particulartherapeutic regiment aimed at ameliorating the disorder is effective.

The materials for use in the method of the invention are ideally suitedfor the preparation of a kit. Such a kit may comprise a carrier meansbeing compartmentalized to receive in close confinement one or morecontainer means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. For example, one of the container means may comprise abinding reagent to any one of the four Leptospira membrane proteins,such as an antibody. A second container may further comprise any one ofthe four Leptospira membrane proteins recognized by such antibody. Theconstituents may be present in liquid or lyophilized form, as desired.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLES

The following examples describe the identification of four Leptospiramembrane proteins i.e. kinase, permease, mannosyltransferase andendoflagellin as important leptospiral proteins that are produced duringactive Leptospiral infection of an animal. The methods by which thesegenes were cloned and sequenced is described. Sequence analysis andhomology studies are shown, further indicating that these proteins aremembrane proteins of pathogenic Leptospira and therefore are excellentvaccine candidates.

Example 1 Bacterial Strains and Growth Conditions

Leptospira interrogans sv pomona strain 2966 and Leptospiraborgpetersenii sv hardjobovis Hb 197 was obtained from freezer stocks ofan original bovine field isolate. All leptospira in vitro cultures werepropagated at 30° C. in PLM-5 broth. Escherichia coli strain DH5a waspropagated in Luria-Bertani medium, with or without 100 μg/mlampicillin, or 50 μg/ml kanamycin. E. coil strain LE392 was utilized inthe construction and amplification of the genomic minibank library. AllE. coli cultures were propagated at 37° C. In some cases (pDFX210, ORF1)expression of heterologous cloned proteins in an E. coil host was doneusing the pL heat shock promoter. Under these circumstances, the strainwas initially propagated at 30° C. in 2× yeast tryptone medium until anoptical density (695 nm) of 0.5 was reached. The culture was shifted to42° C. to induce protein expression.

Animal Passage of L. interrogans sv pomona and L. borgpetersenii svharjobovis Hb197

Animal passage of virulent cultures were done in Syrian hamsters usingliver homogenates of the infected animals as an inoculum for subsequentpassage. A total of 0.2 cc of a 10⁻¹ dilution of infected liverhomogenate in Stuart's Medium or PLM-5 supplemented with 0.1% agarosewas used to inoculate hamsters for passage by subcutaneous (L. pomona)and intraperotoneal (L. hardjobovis) routes of administration.Microscopic examination of liver homogenate smears were done to confirmthe presence of spirochetes.

Extraction and Purification of Bacterial mRNA from Infected HamsterLiver

The Leptospira infected hamsters were euthanised by CO₂ followed bycervical dislocation. The livers were harvested by necropsy and washedtwice with ice cold PBS. The livers were resuspended in 10 ml PBS (roomtemperature) to which an equal volume of 4M guanidine isothiocyanate in50 mM sodium citrate, 0.1% sodium docecyl sulfate was added. Thissuspension was incubated on ice with intermitted vortexing until thetissue was visibly disassociated, water-saturated phenol (pH 5.2) wasimmediately added to facilitate the removal of DNA. This mixture wasvortexed for one minute, then centrifuged at 10,000×g at roomtemperature for 30 minutes. The aqueous phase, which contains thenucleic acids, was extracted twice with TRIS-buffered phenol (pH8.0):chloroform:isoamyl alcohol (25:24:1). This predominantlyRNA-containing aqueous phase was precipitated by the addition of 0.1volume of 3 M sodium acetate (pH 4.5) and 2.5 volumes of 95% ethanol andincubated overnight at −20° C. The precipitated nucleic acids werepelleted and washed. To further facilitate the removal of remaining DNA,the pellet was extracted in 5 ml of 3 M sodium acetate (pH 6.0). Theextraction was repeated until the nucleic acid pellet was transparent.The quality and quantity of the RNA was determinedspectrophotometrically by examining the absorbance ratios at 254 nm/280nm, and at 260 nm/230 nm.

The eukaryotic mRNA was removed from the preparation with an oligo dTcellulose column using the FastTrack mRNA isolation kit (InvitrogenCorporation) with a modification of the manufacturer's instructions.Briefly, the concentration NaCl concentration of the RNA preparation wasadjusted to 0.5 M using 5 M NaCl. the preparation was then added to 50mg of oligo dT cellulose that had been pre-equilibrated with 1 ml of thekit's binding buffer. The mixture was incubated for 60 minutes at roomtemperature with frequent, gentle mixing. Following incubation, theoligo dT cellulose was pelleted at room temperature by centrifugation at2,000×g for 10 minutes. The supernatant, containing the bacterial mRNAwas removed and precipitated by the addition of 0.1 volume sodiumacetate (pH 4.5), 2.5 volumes of 95% ethanol, and incubated overnight at−20° C. The bacterial mRNA was stored in this fashion until needed.

Total bacterial RNA was isolated from PLM-5 propagated strain followingincubation for 96 hours at 30° C. Bacterial cells were removed from theagar by scraping, and washed four times in ice cold PBS. the RNA wasisolated as described above, without the oligo dT cellulose extraction.

cDNA Synthesis

The bacterial mRNA isolated from the infected livers or the PLM-5propagated cultures were used as templates for the synthesis ofdouble-stranded cDNA using avian meoblastosis virus (AMV) reversetranscriptase and the RiboClone cDNA synthesis system (PromegaCorporation, Madison, Wis. USA) following the manufacturer'sinstructions. The newly synthesized cDNA was treated with DNase-freeRNase for 30 minutes at 37° C., extracted once withphenol:chloroform:isoamyl alcohol, and precipitated. The second strandwas synthesized immediately following the first strand synthesisaccording to the manufacturer's instructions.

The cDNA derived from the PLM-5 propagated bacteria was biotinylatedusing the BioNick Labeling System (BRL Life technologies, Gaithersburg,Md.) following the manufacturers' instructions.

In preparation for the subtractive hybridization, the ds cDNApreparations were denatured by incubation at 95° C. for 5 min, followedby rapid chilling in an ice bath.

Subtractive Hybridization

The technique of subtractive hybridization was used to isolate the fourLeptospira genes of the instant invention (Utt, E. A., et al., Can. J.Microbiol. 41:152-156 (1995)). This technique can be used for isolatinga particular mRNA when there are two cell types, one of which expressesthe RNA and the other of which does not. In the present invention, itwas determined that certain Leptospira genes were turned on andexpressed during active infection of this microorganism in a hamstermodel. These four genes are not expressed when Leptospira is grown inculture media. The mRNA from livers of Leptospira infected hamsters(“target or Vir⁺ cells”) is used as substrate to prepare a set of cDNAmolecules corresponding to all the expressed genes. To remove sequencesthat are not specific for the target cells, the cDNA preparation isexhaustively hybridized with the mRNA of Leptospira grown in culturemedia (“Vir⁻ cells”). This step removes all the sequences from the cDNApreparation that are common to the two cell types. After discarding allthe cDNA sequences that hybridize with the other mRNA, those that areleft are hybridized with the mRNA from the target cell to confirm thatthey represent coding sequences. These clones contain sequences specificto the mRNA population of the Vir⁺ cells.

For the subtractive hybridization, a total of 50 μg of denatured in vivobacterial cDNA from the liver extract (Vir+) was hybridized with 250 μgdenatured biotinylated cDNA from the PLM-5 propagated (Vir−) in ahybridization buffer containing 20 mM TRIS, 0.6 M NaCl, 2 mM EDTA, and0.2% sodium dodecyl sulfate (final concentration). The hybridizationproceeded at a constant temperature of 70° C. for 48 hours. The 1:4ratio of the two cDNA populations helped to ensure the formation of cDNAhybrids for all common transcriptional species.

Selective removal of all double-stranded biotinylated cDNA hybrids wasdone by incubation of the hybridization mixture with streptavidin-coatedparamagnetic beads (Dynal, Inc.). A total of 200 μl Dynal streptavidinbeads were placed in a 1.5 ml eppendorf tube and washed 3 times with 2×Dynal binding buffer (10 mM TRIS (pH 7.5), 1 mM EDTA, 2.0 M NaCl. Afterthe final wash the beads were resuspended in 200 μl 2× binding buffer,to which an equal volume of the subtractive hybridization mixture wasadded. This was incubated on a platform rotator at room temperature for15 min at 85 rpm. Following incubation, the paramagnetic bead—boundbiotinylated cDNA hybrids were removed by magnetic extraction accordingto Dynal's instructions. Nucleic acids in the remaining supernatant wereprecipitated by the addition of 0.1 volume 3M sodium acetate (pH 5.3)and 0.8 volume isopropyl alcohol.

The cDNA products that remained in the supernatant putatively representthe differences in gene expression between the liver propagated andPLM-5 propagated L. interrogans. These products are subsequentlyreferred to as subtraction products.

Amplification of the Subtractive Hybridization Products

The double stranded cDNA subtraction products were restrictionendonuclease digested with Sau3A, and then ligated to tandem, 21-mer PCRprimer adapters. The adapters, AUS 1, SEQ ID NO: 9,(5′GATCGGACGGTGAATTCTCGAGAGTG 3′), and AUS 2, SEQ ID NO: 10,(5′GACACTCTCGAGAATTCACCGTCC 3′) both have Sau3A sites, arephosphorylated, and were annealed to each other by heating to 94° C. andcooled to room temperature (25° C.) prior to ligation to the subtractionproducts. The AUS 2 complementary primer was used in the subsequent PCRamplification step. PCR reaction conditions were 10 mM Tris-HCl (pH8.0), 50 mM KCl, 2.5 mM MgCl₂, 10 mM each dNTP, 1 pmole AUS 2, and 10units Taq DNA polymerase (all final concentrations). The total reactionvolume was 100 ml. A Perkin-Elmer Cetus model 9600 thermocycler wasutilized using the following program, with rapid temperature ramping:94° C., 60 sec; 37° C., 30 sec; 55° C., 60 sec; for a total of 35cycles.

Isolation and Identification of Differentially Expressed Genomic Loci

Plasmids pUC18 and pGEMT EASY were used as vectors for cloning into E.coli DH5a. All minibank library constructions, transfections,electroporations, plasmid isolation, restriction endonucleasedigestions, and other genetic manipulations, were done according tostandard methods (Maniatis. et al., 1982).

The PCR subtraction products were directly cloned into the commercialpGEMT—Easy vector (Promega). Positive clones were screened on X-Galampicillin Luria Bertani plates, and the cloned inserts were analyzed byDNA sequencing (Advanced Genetics Analysis Corporation, Minneapolis,Minn.). DNA sequences were identified by sequence database searchesusing the BLAST algorithm. One clone, pHLE001 displayed homology toseveral bacterial membrane bound histidine kinase genes. Since thesubtraction product represented a partial gene, this clone, was labeledwith digoxigenin (DIG) using the Genius System (Boehreinger MannheimBiochemicals, Indianapolis, Ind., USA) according to the manufacturer'sinstructions. The subtraction product was then used as a probe in aSouthern hybridization of EcoRI restriction endonuclease digestedLeptospira interrogans sv pomona (Leptospira pomona) genomic DNA. ThepHLE001 probe hybridized to a 1200 bp fragment. An EcoRI genomicminibank in the 800 bp to 2,500 bp size range was generated and used torecover the 1,200 bp fragment. This fragment was cloned into plasmidpUC18 and was designated pHLE011. This clone was later determined tocontain elements of both ORF1 and ORF2.

Separate protein expression vectors were used to express the identifiedORFs to levels sufficient to small scale protein purification andvaccine protection studies.

Additional cloning was accomplished using the Vectorette strategy(Genosys, Woodlands, Tex.) This strategy was employed to walk throughEcoRI Vectorette genomic libraries and find the remaining portions ofthe genes. Briefly specific primers were designed from the knownsequenced portions to walk both upstream and downstream of the originalPCR product. This strategy resulted in the amplification of fragmentsfrom several subtraction—derived PCR primers. These clones and theirputative identities are summarized in table 1.

DNA sequence analysis of the cloned 1200 bp insert was accomplishedusing the dideoxy chain termination method of Sanger using the AppliedBiosystems automated DNA sequencer.

DNA sequence analysis of the pHLE011 clone using the DNASTAR programrevealed two open reading frames (ORFs). The frames designated ORF1,ORF2 and ORF3 consist of 603 bp, 1131 bp and 618 bp, respectively.

TABLE 1 Subtractive hybridization clone identified in this study. CloneVector Insert Size Sequence pHLE011 pUC18 1,200 bp ORF1/ORF2 pMW43pFLEX10  1110 bp ORF1 pMW310 pFLEX10  1150 bp ORF2 pMW48 pFLEX10   680bp ORF3

Vectorette Library Construction and Screening

Vectorette PCR (Genesys Inc.) following the manufacturers' instructionswas employed in an attempt to isolate the remaining portions of bothORFs. Briefly, PCR primers were designed for the 5′ end of ORF1 toextend upstream. For ORF2, primers were designed for the 3′ end of ORF2to extend downstream. Vectorette libraries were generated using PvuII,HindIII, HpaI, Rsa1, EcoR1, Ssp1, Dra1, or MfeI restriction digestion ofthe L. interrogans genomic DNA, followed by ligation to the specificVectorette primer binding site at one end, according to themanufacturers directions. Vectorette PCR using the above librariesresulted in the generation of several products of various lengths. DNAsequence analysis of the specific products were used to aligned thesequences and allow for the completion of ORF2 and truncated version ofORF1. The subtraction clones are outlined in table 1.

λZAP Phage Genomic Library Construction and Screening

A complete Bm HI genomic library of L. borgpetersenii sv hardjobovis wasconstructed as a custom library package by Stratagene (La Jolla, Calif.,USA) using the ZAP Express vector. It was this library from which theendoflagellin protein was identified.

The library was titered and amplified according to the manufacturersdirections using Escherichia coli XL1-Blue MRF′. Following theappearance of plaques, dry nylon filters (Nytran) which had beenpre-soaked in 5 μM IPTG were placed onto the plates and incubatedinverted overnight at 37° C.

Following overnight incubation, the plates were chilled for one hour at4° C. Filters were then lifted and washed three times in PBST. Thefilters were then blocked by incubation for one hour at room temperaturein PBST/3% non-fat dry milk. After washing the blocked filters threetimes in PBST, immune rabbit antiserum was added to each filter at a1:5,000 dilution in PBST. The primary antibody was incubated with thefilters for three hours at 37° C. Filters were then washed three times,five minutes each, with PBST, and the secondary anti-rabbit alkalinephosphatase conjugate antibody was added at 1:5,000 dilution. Filterswere incubated at 37° C. for two hours. Following incubation with thesecondary antibody, the filters were once with PBST for five minutes,followed by two, five minute washes in PBS. Positive plaques werevisualized by immersing the filters in BCIP solution for one minute atroom temperature.

Isolation and Identification of λZAP Phage Library Clones

A total of fourteen plaques reacted strongly with the immune rabbitantisera. Each of these positive phage were converted to phagemids andtransformed into Eschedchia coli XLOR cells for plasmid isolation andamplification. Each of these clones and their insert size is outlined intable 2.

Positive plaques were excised and converted to phagemids in vivo usingthe method supplied from the manufacturer.

TABLE 2 λZAP Expression library clones identified in this study. CloneVector Insert Size ORF pDFX210 pFLEX10 900 bp endoflagellin

Northern Analysis

Samples of bacterial RNA were analyzed in a Northern hybridization usingthe ³²P-labeled 3.2 kb cloned fragment from pHLE011 as a probe. For theNorthern hybridizations all probes were labeled with ³²P using theMultiprime DNA Labeling Kit (Amersham International Pic, Amersham, UK)following the manufacturer's instructions. Hybridization conditionswere: 5×SSC, 50% formamide, 0.02% SDS, 0.1% N-lauroylsarcosine, 2%sheared salmon sperm DNA, and 20 mM sodium maleate (pH 7.5). Thehybridization proceeded at 42° C. for 16 hours. Stringency washes were:twice with 2×SSC, 0.1% SDS for five minutes at room temperature, andtwice with 0.5×SSC, 0.1% SDS for 15 minutes at 55° C. Signals werevisualized by autoradiography.

DNA Sequence Analysis

Double stranded, bidirectional DNA sequence analysis for selected cloneswas performed using the ABI 200 PRISM System (dye terminator) by LARK(The Woodlands, Tex., USA).

DNA and Primary Sequence Analysis

DNA sequence analysis of the three subtractive hybridization clonesidentified three major open reading frames (ORF). The originalsubtraction clone pHLE011 contained two partial ORFs, designated ORF1and ORF2. Using these partial sequences, Vectorette PCR was used tocomplete both ORF1 and ORF2 sequences, which were subsequently subclonedinto pFLEX10 to yield pMW43 (FIG. 1) and pMW310 (FIG. 2) respectively.DNA sequence analysis of a separate subtraction clone, pHLE004,identified another major ORF designated ORF3. This partial sequence wasused to design PCR primers for Vectorette PCR. This completed the ORF3sequence (FIG. 3).

The primary sequence for all three ORFs was deduced using the DNASTARsoftware package. The deduced primary sequences for the three ORFsidentified putative proteins having molecular weights of 41,000 Da,43,000 Da, and 25,000 Da, respectively. The entire ORF of theendoflagellin gene proved to be 849 bp in length. The deduced primarysequence was a protein of 283 amino acids having a calculated molecularweight of 32,000.

Sequence Database Analysis

A similarity search of these four Leptospira sequences was performedagainst the sequence databases through the National Center forBiotechnology (NCBI) BLAST E-mail server. The BLASTn and BLASTx sequenceanalysis algorithms were employed in an attempt to identify DNA andprimary sequence homologies. Putative gene identities are outlined inTable 3.

TABLE 3 BLAST database homologies of the cloned potential antigensidentified from this study. CLONE GENE HOMOLOGY pMW43 ORF1 membranekinase pMW310 ORF2 membrane permease pMW50 ORF3 mannosyltransferasepDFX210 endoflagellin endoflagellin

Example 2 Syrian Hamster Leptospirosis Challenge Model

Female Syrian hamsters, ranging from one to four months in age, are usedin all bacterial passages and challenges. For the sv pomona challengemodel hamsters are infected by the subcutaneous route using 0.2 ml liverhomogenate containing viable bacteria as determined by phase contrastmicroscopy. Dilution rates of bacteria range from 1:1000 (4×10⁵bacteria/ml) to 1:10 (4×10⁷ bacteria/ml).

For the hardjobovis challenge, hamsters are infected by theintraperotoneal route using 0.5 ml liver homogenate containing viablebacteria as determined by phase contrast microscopy. Dilution rates ofbacteria range from 1:1000 (4×10⁵ bacteria/ml) to 1:10 (4×10⁷bacteria/ml).

Hamster liver tissue containing the viable leptospires are surgicallyexcised following necropsy and processed as follows. Approximately onegram of tissue is placed in a glass dounce homogenizer. A total of ninemilliliters of PLM-5 supplemented with 0.1% agarose is then added to thetissue. The tissue is then homogenized to a uniform consistency. Thisrepresents a 10⁻¹ dilution of bacteria. All subsequent dilutions aremade using the PLM-5 diluent.

Recombinant Protein Expression

Depending upon the expression vector used, proteins were induced usingheat shock for the PL promoter plasmid or IPTG using the lacZ promoterplasmid. All protein expression was done in E. coli DH5α (lacZ) or E.coli LE392(PL) while propagation was done in 2× yeast tryptone broth(2×YT broth).

Briefly, cultures using lacZ expression were propagated at 37° C. untilan optical density (at 695 nm) of 0.4 to 0.5 was achieved. Recombinantproteins were induced by the addition of 1 mM IPTG(isopropylthio-β-D-galactopyranoside). Incubation was continued fromanywhere between 2 to 12 hours, depending upon the expected proteinyield.

Constructs using the PL promoter system were propagated at 30° C. untilan optical density (at 695 nm) of 0.4 to 0.5 was achieved. Recombinantproteins were induced by shifting the culture temperature to 42° C. andcontinuing the incubation for two to four hours. Bacterial cells wereharvested by centrifugation at 8,000×G for 15 minutes at 4° C. Cellswere lysed by two passes through a French pressure cell at 20,000 psi.Bacterial debris was removed by centrifugation at 20,000×G and thesupernatants stored at −20° C. until needed.

Protein extracts were assayed by SDS-PAGE according to standard methods(ref).

Protective Efficacy Data of Experimental Vaccine Antigens in the HamsterLeptospirosis Model

Protein extracts from the recombinant clone containing pHLE011 protected4/6 hamsters (67%) from lethal infection in the sv pomona infectionmodel. Purified recombinant protein from pMW43, contain only ORF1 frompHLE011, protected 2/6 hamsters (33%) from lethal infection in the svpomona infection model. The purified recombinant protein from pDFX210did not provide any protection against the sv pomona infection. Theprotective potential of each of these antigens in the sv hardjobovismodel is underway. Table 4 summarizes the vaccine challenge results forall the antigens tested.

For experimental vaccinations, the animals are vaccinated twice, twoweeks apart, with ca: 5 μg of the experimental protein. At two weeksfollowing the last vaccination, the animals are challenged with eitherserovar using the procedure outlined above.

TABLE 4 Protective potential of selected antigens against lethalleptospira challenge in the Syrian hamster challenge model ofLeptospirosis. Antigen Pomona Hardjobovis clone Source ProtectionProtection pHLE011 subtraction library 67% — pMW43 ORF1 (pHLE011) 33% —pDFX210 phage library 0% 100%

10 1 201 PRT Leptospira interrogans as pomona 1 Met Arg Ser Val Gln GluLys Asn Glu Leu Ile Gln Glu Ile His His 1 5 10 15 Arg Val Arg Asn AsnLeu Gln Val Ile Ser Gly Leu Val Glu Met His 20 25 30 Ser Gly Ser Gly LysGlu Asn Leu Gln Ile Ile Leu Ser Asp Phe Gln 35 40 45 Asn Arg Ile Leu AlaIle Ser Glu Val His Asn Tyr Leu Tyr Lys Ser 50 55 60 Glu Asn Tyr Phe GluIle Asp Phe Val Glu Val Met Asp Lys Ile Ile 65 70 75 80 Leu Asn Leu SerTyr Arg Leu Gly Lys Arg Ser Ile Lys Ile Glu Thr 85 90 95 Glu Ala Glu SerThr Phe Leu Arg Ile Glu Asn Ala Ile Pro Cys Ala 100 105 110 Met Ile PheAsn Glu Leu Leu Ser Asn Ser Leu Lys His Ala Phe Arg 115 120 125 Ser GluLeu Gly Thr Val Gln Ile Ser Phe Arg Lys Lys Gly Asp Lys 130 135 140 TyrTyr Leu Gln Val Ser Asp Asn Gly Ser Gly Ile Lys Asp Phe Lys 145 150 155160 Ile Trp Ser Lys Pro Lys Thr Ala Gly Phe Thr Leu Ile Gln Ile Leu 165170 175 Thr Lys Gln Ile Lys Gly Arg Phe Gln Ile Phe Ser Glu Gly Gly Phe180 185 190 Thr Ala Val Leu Glu Phe Asn Ser Ile 195 200 2 377 PRTLeptospira interrogans sv pomona 2 Met Lys Phe Ser Gly Leu Thr Asn HisIle Tyr Lys Asp Arg Asp Tyr 1 5 10 15 Leu Thr Arg Asn Arg Ala Phe HisLeu Phe Ile Phe Asn Val Val Ser 20 25 30 Leu Leu Leu Gly Leu Ser Val AsnPhe Tyr Val Trp Phe Val Lys Gly 35 40 45 Asp Leu Leu Arg Pro Gly Phe LeuIle Ile Met Leu Ala Ser Ala Val 50 55 60 Ser Leu Phe Phe Leu Leu Arg LysLys Phe Glu Leu Ala Leu Arg Ile 65 70 75 80 Ile Leu Ile Ala Ser Val IleAla Val Ser Val Gly Trp Phe Phe Gly 85 90 95 Leu Ser Gln Gly Asn Ser ProLeu Asp Glu Gly Asn Lys Asn Ile Val 100 105 110 Leu Ala Ile Phe Ile MetIle Phe Leu Tyr Phe Ala Asn Val Lys Arg 115 120 125 Thr Leu Leu Ile AlaVal Tyr Cys Phe Val Leu Ile Phe Met Glu Glu 130 135 140 Leu Leu Met GlnGln Ile His Glu Ser Ile His Met Ala Asp Arg Ile 145 150 155 160 Ala LeuPhe Phe Met Phe Ser Val Ile Ser Ile Ile Ala Val Arg Thr 165 170 175 LeuHis Gly Ser Ile Glu Glu Lys Asn Glu Leu Ile Gln Glu Ile His 180 185 190His Arg Val Arg Asn Asn Leu Gln Val Leu Ser Gly Leu Val Glu Met 195 200205 His Ser Asp Ser Asp Gln Gly Asn Leu Arg Asn Ile Leu Ser Asp Phe 210215 220 Gln Asn Arg Ile Leu Ala Ile Ser Glu Val His Asn Tyr Leu Tyr Lys225 230 235 240 Ser Glu Asn Tyr Phe Asp Ile Asp Phe Ser Glu Val Ile GluArg Ile 245 250 255 Ile Ala Asn Leu Ile His Lys Phe Gly Lys Gln Ser ValLys Ile Glu 260 265 270 Asn Leu Thr Glu Gln Ile Phe Leu Arg Ile Glu TyrAla Ile Pro Cys 275 280 285 Ala Met Ile Phe Ser Glu Leu Leu Ser Asn SerLeu Lys His Ala Phe 290 295 300 Ser Ser Asp Met Gly Lys Ile Val Ile ArgPhe His Lys Glu Gly Asn 305 310 315 320 Lys Tyr Arg Leu Gln Ile Glu AspAsn Gly Ser Gly Ile Ser Asp Ser 325 330 335 Lys Thr Trp Leu Lys Pro LysThr Ser Gly Phe Lys Leu Ile Gln Leu 340 345 350 Leu Thr Arg Gln Ile LysGly Asp Phe Gln Ile Leu Ser Asp Ser Gly 355 360 365 Ser Ile Ala Val LeuGlu Phe Tyr Thr 370 375 3 205 PRT Leptospira interrogans sv pomona 3 MetPhe Asn Phe Ser Gln Tyr Leu Thr Asn Gly Phe Glu Arg Phe Pro 1 5 10 15Leu Ile Glu Lys Ser Lys Ser Lys Ile Lys Lys Ile Ile Phe Val Gly 20 25 30Arg Ile Thr Pro Asn Lys Lys Gln Asp Asp Leu Ile Arg Leu Ala Phe 35 40 45Ala Tyr Lys Ser Ile Ile Ser Asp Gln Phe Gln Phe Tyr Leu Ala Gly 50 55 60Phe Ser Ser Lys Glu Leu Tyr Leu Tyr Arg Glu Glu Leu Glu Arg Met 65 70 7580 Leu Asp Phe Tyr Asp Leu Arg Lys Asn Val Leu Ile Thr Gly Phe Leu 85 9095 Ser Asp Leu Glu Leu Asn Ser Leu Tyr Gln Glu Ala Asp Ala Phe Val 100105 110 Ser Met Ser Glu His Glu Gly Phe Cys Val Pro Leu Ile Glu Ala Met115 120 125 Ile Tyr Arg Ile Pro Ile Leu Ala Phe Ser Gly Gly Ala Val SerGlu 130 135 140 Thr Leu Asn Gly Ala Gly Val Leu Phe Lys Glu Lys Asn PhePro Asn 145 150 155 160 Leu Ala Ile Leu Leu Asn Lys Ile Leu Thr Asp ValSer Phe Gln Asn 165 170 175 Gln Ile Leu Thr Gly Gln Asp Leu Arg Leu AsnGlu Phe Lys Lys Thr 180 185 190 Asp Tyr Lys Ser Val Leu Arg Lys Ala LeuGlu Ile Ile 195 200 205 4 283 PRT Leptospira hardjobovis 4 Met Ile IleAsn His Asn Leu Ser Ala Val Asn Ser His Arg Ser Leu 1 5 10 15 Lys PheAsn Glu Leu Ala Val Asp Lys Thr Met Lys Ala Leu Ser Ser 20 25 30 Gly MetArg Ile Asn Ser Val Ala Asp Asp Ala Phe Gly Leu Ala Val 35 40 45 Ser GluLys Leu Arg Thr Gln Ile Asn Gly Leu Arg Gln Ala Glu Arg 50 55 60 Asn ThrGlu Asp Gly Met Ser Phe Ile Gln Thr Ala Glu Gly Phe Leu 65 70 75 80 GluGln Thr Ser Asn Ile Ile Gln Arg Ile Arg Val Leu Ala Ser Arg 85 90 95 ProArg Met Val Ser Gln Gln Arg Lys Ile Ala Ser Leu Gly Arg Trp 100 105 110Glu Val Leu Cys Ala Gly Gly Pro Lys Ser His Arg Ile Ala Ser Gln 115 120125 Ala Glu Phe Ile Ser Ser Ser Phe Leu Gly Ala Ile Arg Lys Arg Phe 130135 140 Thr Gly Arg Val His Val Val Ser Tyr Gly Ala Glu Arg Lys Ser Ala145 150 155 160 Arg Glu Ile Leu Gln Arg Pro Glu Cys Phe Glu Ser Pro GluAla Cys 165 170 175 Lys Ala Asp Gly Arg Pro Ile Ala Ile Ser Ser Pro GluGlu Ala Asn 180 185 190 Asp Val Ile Gly Leu Ala Asp Ala Ala Leu Thr ArgIle Met Lys Gln 195 200 205 Arg Ala Asp Met Gly Ala Tyr Tyr Asn Arg LeuGlu Tyr Thr Ala Lys 210 215 220 Gly Val Met Gly Ala Tyr Glu Asn Met GlnAla Ser Glu Ser Arg Ile 225 230 235 240 Arg Asp Ala Asp Met Ala Glu GluVal Val Ser Leu Thr Thr Lys Gln 245 250 255 Ile Leu Val Gln Ser Gly ThrAla Met Leu Ala Gln Ala Asn Met Lys 260 265 270 Pro Asn Ser Val Leu LysLeu Leu Gln His Ile 275 280 5 603 DNA Leptospira interrogans sv pomona 5atgcgatccg ttcaagaaaa gaacgaattg atacaagaaa ttcatcatag agttagaaat 60aatcttcagg taatttccgg tttagtggaa atgcatagtg ggtctggtaa agagaatctg 120caaatcatat tatccgattt tcaaaatcgt atattagcaa tatctgaagt tcataattat 180ttatataagt ccgaaaatta tttcgaaatc gattttgtcg aggtgatgga taagattatt 240ctaaatcttt cttatagatt gggaaaacgt tcgatcaaga tagaaactga agctgagtct 300acttttttaa gaatcgaaaa tgcgattcct tgtgctatga ttttcaacga attgttatcc 360aattctttaa aacacgcttt tcgttcggaa aaaggaaccg ttcaaatttc gtttcgaaaa 420aaaggagata aatattacct tcaagtttct gacaatggtt caggaatcaa ggattttaaa 480atttggtcca aaccgaaaac ggctggtttc actttgatac aaatattaac aaaacagatt 540aaaggtcgtt ttcaaatttt ctctgaaggc ggttttactg cggttttaga gttcaactca 600atc 603 6 1131 DNA Leptospira interrogans sv pomona 6 atgaaattttcaggattaac caatcatatt tataaagaca gggattatct tactcgaaat 60 agggcgttccatcttttcat ttttaatgtg gtgtcgcttt tattgggttt atctgtgaat 120 ttttatgtttggtttgtgaa aggtgatcta ttacgtcctg gttttttaat catcatgctt 180 gcatctgcagtctctctgtt ttttttattg agaaaaaaat ttgaattggc tctcagaatt 240 attttgatcgcaagtgtaat tgctgttagc gttggttggt tttttggact ttctcaggga 300 aattctcctttggacgaagg gaataaaaat attgttttag ctatatttat tatgattttc 360 ttatattttgcaaatgtaaa gcgaactctt ctaattgcgg tttactgttt tgttttgatt 420 tttatggaagagcttttaat gcaacaaatt catgaatcta ttcacatggc tgatcgaatc 480 gctctatttttcatgttttc tgtaatttcg attatcgccg taagaactct tcatggatcg 540 attgaagaaaagaacgaatt gatacaagaa attcatcata gagttagaaa taatcttcag 600 gttctttccggtttagtaga aatgcatagt gattctgatc aagggaatct taggaatata 660 ttatctgattttcaaaatcg tatattagca atatctgaag ttcataatta tttatataag 720 tccgaaaattatttcgacat agatttttca gaagtgattg aaagaatcat tgcaaatctc 780 attcataaatttggtaaaca atctgtaaaa atagaaaatt taacggaaca gattttttta 840 agaatcgaatatgcgattcc ttgtgctatg atttttagtg aacttttatc taattcttta 900 aaacatgcgttttcttcgga tatggggaaa attgtcattc ggtttcataa agaaggaaat 960 aagtatcgtcttcaaattga agataatggt tctggaatat ctgattctaa aacttggttg 1020 aaaccaaaaacttctggttt taaattgatt caacttttga ccagacaaat aaaaggtgat 1080 tttcaaattctttcggattc tggttccatt gctgtacttg aattttacac t 1131 7 618 DNA Leptospirainterrogans sv pomona 7 atgtttaatt tttcccaata cctaaccaat ggttttgaacgttttcctaa aatcgaaaaa 60 tcaaaatcta aaattaaaaa aattattttt gtaggtaggatcactcccaa taaaaaacag 120 gacgatttga tccgccttgc attcgcgtat aagtctataatttccgatca gtttcagttt 180 tatctcgcag gttttagttc taaagaatta tatctttatcgggaagaatt agaaaggatg 240 ttggactttt atgatctcag aaaaaacgtt ttgatcacaggttttctctc cgacttagaa 300 ctaaattccc tttatcaaga agcggatgct ttcgtttccatgagtgaaca cgaaggtttc 360 tgtgttcctc tgatcgaagc catgatttat agaattccgatcctcgcttt ttcaggcggc 420 gcggtttccg aaactttaaa cggagccggt gttctttttaaagaaaaaaa ttttccgaac 480 ttggctattt tactcaataa aattttgact gatgtttctttccaaaatca aattttaaca 540 ggccaagatc tacgtctgaa cgaatttaaa aaaacggattataaatccgt ccttaggaag 600 gcacttgaaa tcatctct 618 8 849 DNA Leptospirahardjobovis 8 atgattatca atcacaacct gagcgcggtg aattctcacc gttctctaaagttcaacgag 60 cttgctgtgg acaagacgat gaaggctttg tcttccggta tgcggatcaattccgtggcg 120 gacgacgctt tcggactcgc ggtttctgaa aagctaagaa cgcagatcaacggtctgcgt 180 caggccgaaa gaaacaccga agacgggatg agcttcattc aaactgccgagggtttcctc 240 gaacagacgt cgaacatcat tcagagaatc cgggtgcttg catccagacctcgaatggtt 300 tctcagcaac gaaagattgc atctttgggc aggtgggaag tattgtgcgctggtggacca 360 aagtcccacc gaatcgcttc tcaagctgaa tttataagtt caagctttttaggggcaatt 420 cgcaaaaggt tcacgggtcg ggtccatgtg gtttcatatg gggccgaacgaaaatcagcg 480 agagagattt tacagcggcc cgaatgcttc gaaagccctg aagcttgtaaagcggacggg 540 agaccgatcg cgatttcttc tccggaagaa gccaacgatg ttatcggtttagcggatgcg 600 gctcttacga ggatcatgaa gcagagagcg gatatggggg cttattacaataggctcgag 660 tataccgcaa aaggggtgat gggtgcatat gaaaatatgc aagcatcggaatccagaatt 720 cgggacgccg atatggcgga ggaagttgtc tcgctgacca caaaacaaatactcgttcag 780 agtggtacgg caatgttagc gcaggcaaat atgaaaccga attcggttctcaagcttctg 840 cagcatatc 849 9 26 DNA Artificial Sequence Primer 9gatcggacgg tgaattctcg agagtg 26 10 24 DNA Artificial Sequence Primer 10gacactctcg agaattcacc gtcc 24

What is claimed is:
 1. An isolated protein of Leptospira interrogans svpomona comprising the amino acid sequence of SEQ ID NO:2.
 2. Apharmaceutical composition for inducing an immune response to pathogenicLeptospira interrogans sv Pomona in an animal comprising animmunogenically effective amount of the isolated protein of claim 2 anda pharmaceutically acceptable carrier.